Although the plant constituents of livestock feedstuffs are rich in phosphorus, inorganic phosphorus supplementation is required to obtain good growth performance of monogastric animals. Phytic acid (myo-inositol hexaphosphoric acid) generally occurs as a complex of calcium, magnesium and potassium salts and/or proteins, and is the predominant form of phosphorus in cereals, oil seeds, and legumes, and accounts for 1 to 3% of the seed dry weight and 60 to 90% of the total phosphorus present in seeds (Graf, 1986). However, monogastric animals (e.g., swine, poultry and fish) utilize phytate poorly or not at all because they are deficient in gastrointestinal tract enzymes capable of hydrolyzing phytate. Phytate passes largely intact through the upper gastrointestinal tract, where it may decrease the bioavailability of nutrients by chelating minerals (e.g., calcium and zinc), binding amino acids and proteins (Graf, 1986) and inhibiting enzymes. Phytate phosphorus in manure poses a serious pollution problem, contributing to eutrophication of surface waters in areas of the world where monogastric livestock production is intensive.
Production inefficiencies and phosphorus pollution caused by phytate may be effectively addressed by phytase supplementation of diets for monogastric animals. Phytases catalyze the hydrolysis of phytate to myo-inositol and inorganic phosphate, which are then absorbed in the small intestine. In addition to decreasing phosphorus supplementation requirements and reducing the amount of phytate pollutants released, phytases also diminish the antinutritional effects of phytate.
Phytases are produced in animal and plant (predominantly seeds) tissues and by a variety of microorganisms (U.S. Pat. No. 3,297,548; Shieh and Ware, 1968; Ware and Shieh, 1967). Despite the array of potential phytase sources, only soil fungi (Aspergillus niger or Aspergillus ficuum) are currently used for commercial production of phytase. The phytase produced by A. ficuum possesses greater specific activity (100 units/mg of protein (wherein units are defined as Imoles of phosphate released per minute)) and thermostability compared to those phytases that have been characterized from other microorganisms (European Patent Application No. 0,420,358 van Gorcum et al., 1991 and U.S. Pat. No. 5,436,156 (van Gorcum et al., issued Jul. 25, 1995)). The A. ficuum phytase is an acid phytase and exhibits little activity above pH 5.5 (Howson and Davis, 1983; van Gorcum et al., 1991). Consequently, activity is limited to a relatively small region of the monogastric digestive tract, in which the pH ranges from 2-3 (in the stomach) to 4-7 (in the small intestine).
Although the idea of phytase supplementation of monogastric diets was proposed more than 25 years ago (U.S. Pat. No. 3,297,548, Ware and Shieh, 1967), the high cost of enzyme production has restricted the use of phytase in the livestock industry. In North America, supplemental phytase is generally more expensive than phosphorus supplements. In some circumstances, the cost of phytase utilization may be partially offset if the use of this enzyme also decreases the need for supplementation of a second nutrient such as calcium. The use of phytase in North America is likely to increase as swine and poultry populations increase and as public pressures force a reduction in pollution associated with livestock production. Higher costs of phosphorus supplements and legislation requiring the use of phytase have made the use of this supplement more common in Europe and parts of the Orient than in North America. Governments of the Netherlands, Germany, Korea and Taiwan have enacted or are enacting legislation to reduce the phosphorus pollution created by monogastric livestock production.
A more effective means of increasing phytase utilization is through cost reduction. The cost of phytase can be reduced by decreasing production costs and/or producing an enzyme with superior activity. Recent advances in biotechnology may revolutionize the commercial enzyme industry by offering alternative, cost effective methods of enzyme production. Application of recombinant DNA technology has enabled manufacturers to increase the yields and efficiency of enzyme production, and to create new products. The original source organism need no longer limit the production of commercial enzymes. Genes encoding superior enzymes can be transferred from organisms such as anaerobic bacteria and fungi, typically impractical for commercial production, into well characterized industrial microbial production hosts (e.g., Aspergillus and Bacillus spp.). As well, these genes may be transferred to novel plant and animal expression systems.
Unlike monogastric animals, ruminants (e.g., cattle, sheep) readily utilize the phosphorus in phytic acid. It has been demonstrated that phytases are present in the rumen, and it has been proposed that ruminants reared on high grain diets (rich in phytate) do not require dietary phosphorus supplementation due to these ruminal phytases. A single report has attributed this phytase production to ruminal microorganisms (Raun et al., 1956), but overall, the unique capacity of ruminants to utilize phytate has largely been ignored. Raun et al. (1956) prepared microbial suspensions by centrifugal sedimentation (Cheng et al., 1955). Those microbial suspensions were almost certainly contaminated with microscopic particles of plant material. Since plants produce phytases, the study was inconclusive as to whether plant phytases or microbial phytases produced the observed activity. Although Raun et al. have raised the possibility that ruminal phytase production may be attributable to ruminal microorganisms, this possibility has not been explored.
In view of the foregoing, there remains a need for low cost phytases having biochemical characteristics well suited for use in animal feed supplements.